Figure 1.

Pulse sequences used to record steady-state ¹⁵N-{¹H} nuclear Overhauser effects. For each measurement, reference and steady-state experiments were recorded in an interleaved manner. The reference experiment is shown in (a). At the end of the recovery delay T = 10 s, the proton carrier is placed on resonance with the water signal and a very selective water-flip-back pulse is applied (3 ms sinc shaped). To record steady-state experiments, the boxed sequence in (a) was substituted by the schemes shown in (b-e) for the effective saturation of amide proton resonances. The recommended effective saturation scheme is shown in (b): after an optional delay T′ = 2 s for stable detection of the lock signal, the proton carrier is placed at the center of the amide region (at 8.2 ppm) as shown by the arrow labeled N. The motif [delay τ/2 – 180° pulse - delay τ/2] is repeated n times. The interpulse delay τ is typically 22 ms (11 ms may also be used, see text). The RF amplitude for the pulses was 7.5 kHz at 500 MHz Larmor frequency and 9 kHz at 600 MHz. A gradient G₁ is applied at the end of the last τ/2 delay to suppress all transverse components of the proton polarization. The carrier was then moved on-resonance with the water signal as indicated by the W arrow. To evaluate the influence of several effects the scheme (c) was used. The carrier was set to frequency F during proton irradiation, a delay τ of 5 ms or 22 ms was used and pulses with variable tilt angles β were employed. (d) Same irradiation scheme as in (c) was used, except that the square RF pulses were replaced by Broadband inversion pulses (BIP) [17] with durations of 72 μs and RF amplitudes of 18 kHz. (e) Composite pulse decoupling (CPD) with GARP [19] and WALTZ-16 scheme was used for proton effective saturation with a duration T″ = 4 s and RF amplitudes of 1 or 1.2 kHz (see text). Similarly, when schemes b-d were used, the number of cycles n was always set so that the total duration for effective saturation was 4 s. All narrow (filled) and (wide) open rectangles represent 90° and 180° pulses respectively. Pulse phases are along the x-axis of the rotating frame unless otherwise mentioned. Proton composite pulse decoupling during the delay t₁ was performed with a GARP scheme and an RF amplitude of 1 kHz. Composite-pulse decoupling during acquisition was performed on the ¹⁵N channel with a GARP scheme and an RF amplitude of 1090 Hz. The delay τ_a was set to 2.56 ms. The phases were: φ₁ = {y, -y}; φ₂ = {x, x, -x, -x}; φ₃ = {x, x, -x, -x}; φ₄ = {-y, -y, y, y}; φ_acq = {x, -x, -x, x}. The amplitude profile of pulsed-field gradient was a sine bell shape. At 600 MHz, their durations and peak amplitudes over the x, y and z directions were, respectively: G₁; 600 ms, 15 G.cm^-1, 15 G.cm^-1, 0; G₂; 1 ms, 0, 0, 25 G.cm^-1; G₃; 1 ms, 0, 0, 40 G.cm^-1; G₄; 1 ms, 0, 0, 8.1 G.cm^-1 . At 500 MHz, their durations and peak amplitudes along the z direction were, respectively: G₁; 500 ms, 18 G.cm^-1; G₂; 1 ms, 25 G.cm^-1; G₃; 1 ms, 40 G.cm^-1; G₄; 1 ms, 8.1 G.cm^-1 . The carrier was set at 117 ppm on the ¹⁵N channel. The duration of acquisition was 90 ms at 600 MHz and 100 ms at 500 MHz. In the indirect dimension, 64 complex points were recorded with a spectral width of 24 ppm. Coherence selection was achieved by inverting the amplitude of the gradient G₃ and phase φ₄.